Methods for improved targeting of antibody, antibody fragments, hormones and other targeting agents, and conjugates thereof

ABSTRACT

Methods for improved targeting of antibody, antibody fragments, peptides hormones, steroid hormones and conjugates thereof are disclosed. Enhanced delivery to target cells of antibodies or fragments thereof or other receptor-mediated delivery system, such as peptide, specific for a population of cells of a mammal comprises steps of administering to said mammal an adequate dosage of blocking antibodies or fragments thereof or other receptor-mediated delivery system, such as peptide, and administering to said mammal an effective dosage of said antibodies or fragments thereof or other receptor-mediated delivery system, such as peptide, specific for said population of cells. In the preferred embodiment, the specific antibodies are monoclonal antibodies directed toward tumor-associated antigen in man.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of co-pending applicationSer. No. 917,176, filed Oct. 9, 1986 now abandoned.

1. Technical Field

The present invention generally relates to methods for enhancingtargeting of antibodies, antibody fragments, peptide hormones andsteroid hormones, and conjugates thereof. More specifically, methods aredisclosed employing blocking antibodies, fragments, hormones and othertargeting agents, and conjugates thereof to reduce cross-reactive andnonspecific binding of specific antibodies, hormones and other targetingagents to non-target cells.

2. Background Art

Antibodies are proteins that have a binding site that is specific for aparticular determinant, e.g., antigen or epitope, and other portionsthat bind to normal tissues in a nonspecific fashion. There are severalimmunological concepts, all related to antibody binding, that requiredefinition.

Target-specific binding: Binding of the antibody, whole or fragment,hormones, other targeting agents or conjugate thereof, through theantibody's binding site, to the epitope recognized by said antibody oncells expressing said epitope's or hormone's receptor, where said cellsare the desired target of the antibody, whole or fragment, or hormone,other targeting agent, or conjugates thereof.

An example of target-specific binding is binding of the antibody, wholeor fragment, or conjugate thereof, to tumor cells where the antibody inquestion can also bind specifically to normal cells. The component ofbinding to the tumor cells is target-specific. Another example isbinding of bombesin, or gastrin-releasing peptide, to small cell lungcarcinoma.

Cross-reactive binding: Binding of the antibody, whole or fragment, orhormone, other targeting agent or conjugate thereof, through theantibody's binding site, to the epitope recognized by said antibody oncells expressing said epitope or hormone receptor, where said cells arenot the desired target of the antibody, whole or fragment, or hormone orconjugate thereof.

An example of cross-specific binding is binding of the antibody, wholeor fragment, or conjugate thereof, to normal lung by antibody bindingsite to the same or structurally homologous epitope as is present on atumor cell. The component of binding to the normal lung cells by theantigen-binding site of the antibody is cross-reactive. Another exampleis the binding of bombesin, or gastrin-releasing peptide, to normalcells in the stomach or pancreas.

Nonspecific binding: Binding of an antibody, whole or fragment, orhormone or conjugate thereof, through some mechanism other than theantigen-recognition binding site of the antibody or hormone, to cellsother than the target cells.

An example of nonspecific binding is the uptake of antibody into theliver and spleen due to binding of the antibody by its Fc receptors ontocells in these organs.

A second example would be binding of mannose present in the ricin ofantibody-ricin conjugate to mannose receptors on liver cells.

Specific antibody: Antibody that binds to epitope on desired targetcells through its antigen-recognition sites. Specific antibodies mayalso bind to epitope or structural homolog present on non-target cells.

Irrelevant antibody: Antibody that does not bind to target cells bymeans of its antigen-recognition sites, but may bind to non-target andtarget cells through non-specific mechanisms, e.g., Fc portion ofantibody binding to Fc receptors on cells in reticuloendothelial system(RES).

Blocking antibody: Antibody that inhibits the nonspecific binding ofpharmaceutically active specific antibody. Blocking antibodies mayinclude irrelevant antibody or pharmaceutically inactive specificantibody or fragments or combinations thereof. The latter may also becalled "cold-specific" antibody.

Pharmaceutically active antibody: Anbtibody that is diagnostically ortherapeutically effective.

The use of antibodies as carriers for radionuclides and cytotoxins hasbeen a goal of cancer diagnosis and treatment since Pressman et al.showed that 131_(I) labeled rabbit anti-rat kidney antibodies localizedin the kidney after intravenous injection (Pressman, D., Keighly, G.(1948) J. Immunol. 59:141-46). Just a few years later, Vial and Callahanreported a dramatic, complete response in a patient with widelymetastatic malignant melamoma treated with ¹³¹ I-antibodies raisedagainst his own tumor (Vial, A. B. and Callahan, W. (1956), Univ. Mich.Med. Bull. 20: 284-86). In the 1960s, Bale and co-workers demonstratedthat labeled antibodies to fibrin, which is often deposited in rapidlygrowing tumors, could localize in rat, dog and human tumors (65% of 141patients) (Bale, W. F., et al. (1960) Cancer Res. 20:1501-1504 (1960);McCradle, R. J., Harper, P. V., Spar, I. L., et al. (1966) J. Nucl. Med.7:837-44; Spar, I. L., Bale, W. F., Manack, D., et al. (1969) Cancer78:731- 59; Bale, W. F., Centreras, M. A., Goody, E. D. (1980) CancerRes. 40:3965-2972). Several years later, Chao et al. demonstratedselective uptake of antibody fragments in tumors (Chao, H. F., Peiper,S. C., Philpott, G. W., et al. (1974) Res. Comm. in Chem. Path. & Pharm.9:749-61).

Monoclonal antibodies (Kohler, G. and Milstein, C. (1975) Nature256:495-97) offer advantages over the polyclonals used in these studiesbecause of their improved specificity, purity and consistency amonglots. These factors, plus their wide availability, have led to improvedclinical applications of antibodies and their conjugates.

Even with monoclonals, however, most antibodies to human tumors havesome normal tissue cross-reactivity. Compared to tumors, thesecross-reactive sites may equally or preferentially bind injectedantibody or conjugate and thus adsorb a substantial portion of theadministered dose, especially if these sites are concentrated inwell-perfused organs. If the antibody is conjugated to a toxic agent,there may be toxicity to the normal tissues that could be dose-limiting.Therefore, reducing normal tissue binding of the antibody or conjugateswithout adversely affecting their tumor localization would beadvantageous.

Also needed in the art are methods to improve the targeting ofimmunoconjugates to tumor cells. Most immunoconjugates are produced bychemically linking an antibody to another agent. Another possibility iscreating a fusion protein. The antibody itself, the process of linking,or the conjugated agent itself may cause decreased locatization to thetumor due to nonspecific or cross-reactive binding.

Also needed in the art is a method to improve delivery of cytotoxins orbiologic response modifiers (BRMs) to tumors using antibodies ascarriers, while minimizing toxicity.

Also needed in the art are similar methods to improve delivery ofcytotoxins or biologic response modifiers (BRMs) to tumors usinghormones or other targeting agents as carriers, while minimizingtoxicity.

Also needed in the art is a method to decrease formation ofantiglobulins to injected antibody or immunoconjugates as there may beinhibition of binding or even toxicity when the same antibody isinjected at a later time.

All of these issues can be addressed by the described methods thatreduce cross-reactive and nonspecific binding of antibody and antibodyconjugates, and are equally applicable to any substance that has anonspecific uptake site or whose receptors are shared by non-targetcells.

The methods of the present invention reduce cross-reaction and/ornonspecific binding of specific antibodies and hormones whenadministered to diagnose, stage, evaluate or treat diseases such ascancer in humans. The characteristic of specificity makes antibodiespotentially useful agents for targeting defined populations of cellssuch as tumor cells that express tumor-specific (expressed uniquely bytumor cells) or tumor-associated (expressed by tumor cells and by asubpopulation of normal cells) antigens. The clinical utility of thesespecific antibodies, however, is compromised by the phenomenon ofcross-reactive and nonspecific binding.

One method of markedly diminishing nonspecific uptake is to remove anonspecific binding portion of the antibody, leaving the antigen bindingportion [e.g., F(ab)'_(2'), Fab', Fab or Fv]. Fragments may accumulatemore rapidly onto the tumor cell than whole antibody due to theirsmaller size, which facilitates egress from the circulation across bloodvessel and capillary walls into the tumor bed. This does not usuallycompensate for the decreased serum half-life of the fragments resultingin decreased tumor accumulation compared to whole antibody. Thus thereexists a need to prolong serum half-life of fragments in order to takeadvantage of their more rapid accumulation in target cells and,therefore, to improve localization into a tumor. Additionally,nonspecific binding of specific antibodies into normal organs needs tobe decreased.

Generally, a major obstacle to the successful clinical use of antibodiesand conjugates thereof has been inadequate delivery to target cells.This has been assessed both by immunohistochemistry on frozen sectionsof tumors removed after antibody administration (Oldham, R. K., Foon, K.A., Morgan, A. C., et al. (1984) J. of Chem. Oncol. 2(11):1235-44;Abrams, P. G., Morgan, A. C., Schroff, C. S. (1985) In: MonoclonalAntibodies and Cancer Therapy (Deisfeld and Sell, Eds.), Alan R. LissInc., pp. 233-36) or by calculating the percent of the radiolabeledantibody dose per gram in the tumor (Murray, J. L., Rosenblum, M. G.,Sobol, R. E., et al. (1985) Cancer Res. 45: 2376-81; Carrasquillo, J.A., Abrams, P. G., Schroff, R. W., et al. (submitted); Epenetos, A. A.,Mather, S., Gwanowska, M., et al. (1982) Lancet II:999-1004; Larson, S.M., Carrasquillo, J. A., Krohn, K. A., et al. (1983) J. Clin.Investigation 72:2101-2114 (1983). There was clear evidence in theformer studies of increased antibody localization in tumor with higherdoses of specific antibody. The quantitative studies with radiolabeledantibody have shown 0.0001%-0.0004% of the injected dose/gram localizingto tumor (Carrasquillo, ibid.).

Radiolabeling of antibodies permits the quantitative assessment ofpercent accretion into tumors and normal organs and disappearance fromthe blood and the whole body. This serves as a paradigm for thebiodistribution and kinetics of antibodies and immunoconjugates.Conjugated or labeled peptides or steroid hormones provide an analogousstrategy.

Studies with radiolabeled antibodies have demonstrated that part of theproblem causing low tumor accumulation is the localization ofradiolabeled antibody in other organs, such as liver, spleen, marrow,lung or kidney. The prior art is replete with examples of increasingmass of specific antibody to improve tumor localization (e.g., Abrams,ibid.; Murray, ibid.; Carrasquillo, ibid.; Epenetos, ibid.; Larson,ibid.). The present invention uses irrelevant antibody to adsorb tothese nonspecific sites, thus obviating the requirement for large dosesof unconjugated specific antibody required in the prior art. Oneadvantage of this strategy is that unconjugated irrelevant antibody doesnot compete for specific sites on target cells with conjugated specificantibody. Another advantage is that irrelevant antibody skews theimmunological response of the patient away from the specific antibody(vide infra).

One measure of decreased adsorption of specific antibody ontononspecific sites is prolonged serum half-life of the antibody. Anotherunexpected result of pre-administering isotype and subclass matchedwhole irrelevant immunoglobulin is the prolonged serum half-life,reduced nonspecific adsorption and improved tumor detection withfragments of specific antibody.

The prior art recognizes the problem of localizing specific antibody andfragments and conjugates thereof due to nonspecific and cross-reactivebinding to non-target epitopes, e.g., Murray, ibid. Yet, the solutionemployed has been to lump together the problems of nonspecific andcross-reactive uptake and to use a single strategy ----co-administration of large masses of specific unconjugated antibody ----to overcome the problems. The present invention not only distinguishesthese problems, but also offers different solutions to each: (1) the useof irrelevant antibody (immunoglobulin) to reduce nonspecific andcross-reactive binding of specific antibody, and (2) the administrationof unconjugated specific antibody to bind to cross-reactive sites priorto the administration of conjugated specific antibody. The lattersolution applies equally to steroid and piptide hormones.

Since antibodies are proteins, in most cases of non-human origin, thespectre of human antiglobulin and anti-idotype has been raised (Oldham,ibid.; Abrams, ibid.; Murray, ibid.; Carrasquillo, ibid.; Epenetos,ibid.; Larson, ibid.). This invention demonstrates that theco-administration of larger masses of irrelevant antibody can skew thatantiglobulin response toward the irrelevant antibody and not thespecific antibody so that, along with either the same or a secondirrelevant antibody, the same target-specific antibody can be injectedagain and localize in tumor sites without significant formation ofantiglobulins to the specific antibody.

Despite their increased specifity, monoclonal antibodies thus far havenot been entirely tumor-specific, but rather recognize tumor-associatedantigens. When their cross-reactivity is predominantly to one organ thatcan be selectively perfused, the present invention provides methods fortargeting specific antibody conjugates by selective direct infusion ofunconjugated specific antibody into a vessel feeding a normal organknown to concentrate conjugated antibody by cross-reactive binding. Fora therapeutic conjugate, the reduction in toxicity is a substantialadvantage.

Conjugates of specific antibody may localize in nonspecific sites due tothe molecule linked to the antibody rather than the antibody itself. Theprotein toxins (e.g., ricin, abrin, diphtheria toxin, pseudomonas toxin)can bind to mammalian cells through particular portions of thesemolecules. This property has prompted a large effort to eliminate theirnonspecific binding capability while preserving the potency of thetoxin. According to the methods of the present invention, detoxifiedprotein toxins, when conjugated to nonspecific antibody, can reducebinding of the specific antibody-toxin conjugate to nonspecific sites.This methodology reduces whole organism toxicity and permits theadministration of larger doses of the specific toxin conjugate.

DISCLOSURE OF THE INVENTION

The method of the present invention is for enhancement of delivery totarget cells of antibodies or fragments thereof or otherreceptor-mediated delivery systems, such as peptide, specific for apopulation of cells of a mammal. The method comprises the steps ofadministering to said mammal an adequate dosage of blocking antibodiesor fragments thereof or other receptor-mediated delivery systems, suchas peptide, and administering to said mammal an effective dosage of saidantibodies or fragments thereof or other agents that target a definedpopulation of cells via receptors, such as hormones, specific for saidpopulation of cells, the blocking antibodies or fragments thereof orother targeting agents capable of nonspecific and/or cross-reactivebinding to non-target cells. The antibody fragments of either theblocking antibodies or the specific antibodies are selected from thegroup consisting of F(ab)', F(ab)'₂, Fab, Fv and mixtures thereof.

In a preferred embodiment, the target cells are characterized by havingtumor-associated antigen. Both the blocking antibodies and the specificantibodies may be either monoclonal or polyclonal antibodies.Furthermore, the specific antibodies and fragments thereof may beconjugated to cytotoxins, radionuclides or biological responsemodifiers.

The administration of the blocking antibodies is preferably done priorto the administration of the specific antibodies; alternatively, suchblocking antibodies may be administered simultaneously with the specificantibody. The effective dosage of the antibodies or fragments thereofspecific for a said population of cells is either diagnosticallyeffective or therapeutically effective. The preferred mammal of themethods disclosed herein is man.

An additional preferred embodiment of the methods disclosed herein isfor enhancement of the localization of antibodies or fragments thereofspecific for a cross-reactive antigen contained within a mammal's tissueor organ. This method comprises the steps of directly perfusing saidtissue or organ with an adequate dosage of blocking antibodies orfragments thereof and then administering systemically to the mammal aneffective dosage of said antibodies or fragments thereof specific forsaid antigen contained within said tissue or organ that is also presenton target cells.

A related aspect of the present invention discloses a method forreducing within a mammal the production of anti-immunoglobulin directedagainst antibodies or fragments thereof specific for a population oftarget cells. This method comprises the steps of administering to saidmammal in adequate dosage of blocking antibodies or fragments thereofcapable of stimulating the production of anti-immunoglobulin directedagainst said blocking antibodies or fragments thereof and administeringto said mammal a therapeutically effective dosage of said antibodies orfragments thereof specific for said target cells, wherein saidtherapeutically effective dosage is smaller than said adequate dosage ofblocking antibodies or fragments thereof.

BEST MODE FOR CARRYING OUT THE INVENTION 1. Reducing Nonspecific Uptake

An irrelevant antibody may be matched with a specific antibody byspecies, isotype and/or subclass and is administered, usually inseveral-fold or greater quantities, to patients prior to the injectionof the specific antibody or specific antibody conjugates. Alternatively,such matching may not be necessary. The prior art has demonstratedimproved tumor localization of whole antibody with increasing doses ofspecific antibody. These results were assessed with either trace-labeledspecific antibody or by immunohistochemistry following antibodyadminstration (Oldham, ibid.; Abrams, ibid.; Murray, ibid.;Carrasquillo, ibid.). In the former, the unlabeled, specific antibodywas usually given simultaneously with the labeled preparation. Theunderlying concept involved providing sufficient mass of antibody toattach to nonspecific sites, leaving more specific antibody available tobind to tumor. There was some reduction of labeled antibody innonspecific sites (e.g., liver), and a marked increase in serumhalf-life of the radiolabeled whole immunoglobulin (Carrasquillo,ibid.). There is, however, the potential for competition for specificbinding at the tumor site by the labeled and unlabeled antibody. If thelabel is used symbolically to represent any agent conjugated toantibody, competition of unlabeled antibody for the tumor antigen at themost accessible (perivascular) sites will compromise the delivery ofthat agent to the tumor. In addition, a percentage of the labeledantibody, when injected simultaneously, behaves as a true tracer anddeposits in normal nonspecific and cross-reactived sites.

The method described herein uses irrelevant antibody (blocking antibody)to reduce the nonspecific uptake, without competing for the tumorantigen, preferably administered prior to specific antibody (see ExampleI). The positive results observed by pre-administering irrelevantantibody were not expected since human gammaglobulins, in large (>1 gm)quantities, failed to show any decreased localization of radiolabeledantibody into nonspecific sites (Carrasquillo, ibid.).

Further, the method described herein is applicable to fragments ofspecific antibody as well as to whole specific antibody. The method alsohas been shown to increase the serum half-life of fragments.

Particularly unexpected was the prolonged serum half-life in the initialphase of serum clearance achieved by using whole irrelevant antibodyprior to specific antibody fragments. This was surprising since it wasassumed that the nonspecific binding sites on antibody would beeliminated by creating fragments devoid of the Fc portion of themolecule. This method is useful for reducing non-specific binding ofspecific antibody, whole or fragment, or conjugates of such antibody,whole or fragment, with radionuclides, drugs, toxins, biologic responsemodifiers or differentiating agents.

2. Reducing Cross-Reactivity with Normal Tissues

Normal tissues may contain an epitope, or structural homolog of anepitope, expressed on the target cell. Several methods to reducelocalization to non-target tissues are presented herein. For normaltissues equally or more accessible than tumors to specific antibody,unconjugated specific antibody (blocking antibody) is administered priorto conjugated specific antibody (see Example II). The antigen sites onnormal tissues, such as blood vessels or liver, will first bind thepreviously injected, unconjugated whole antibody. In one embodiment, theblocking antibody is bivalent (whole or F(ab'₂) and the conjugatedantibody is monovalent (Fab', Fab or Fv). This approach taken advantageof the higher antigen affinity of the bivalent molecules and thusreduces the competition of the subsequently administered, conjugatedmonovalent species for the cross-reactive sites.

The precise doses of the cold-specific antibody and the timing ofadministration will be dependent upon the size of the patient, thequantitative normal tissue expression of the antigen or epitope, therelative accessibility of tumor compared to normal tissue sites, and thepurpose for which the entire procedure is being performed. For example,a modest degree of nontumor targeting may be acceptable in a therapeuticstudy so long as toxicity is tolerable, but less acceptable in animaging mode where false positives could be a problem.

The dose of the cold-specific antibody can be approximated byadministering increasing doses of the cold-specific antibody prior tobiodistribution studies with a labeled antibody or labeled conjugate.When normal tissue sites that show evidence of uptake of theradiolabeled antibody or conjugate with no cold-specific antibody are nolonger visualized, than an adequate dose of the cold-specific antibodywill have been reached. Alternatively, a greater interval may be allowedbetween the given dose of cold-specific antibody and the subsequentadministration of conjugated specific antibody to permit greaterpermeation of the cold-specific antibody into normal tissues.

Another approach is to determine the serum half-life of an irrelevantantibody or fragment and preadminister sufficient cold-specific antibodyor fragment to raise the half-life of the conjugated antibody orfragment close to the half-life of the irrelevant antibody. This wouldbe indirect evidence that a normal tissue antigen "sink" was completelyor largely saturated.

An additional method of determining the appropriate dose has beendescribed by Eger et al. Using data obtained from patients receiving the9.2.27 antibody, they constructed a mathematical model that, byregression analysis, could predict a dose of antibody that wouldsaturate the "antigen sink" in normal tissues.

Once determined for a particular antigen-antibody system, the dose andschedule can be calculated on a body surface area or weight basis forthe particular antibody.

Within a wide range, however, the administration of more than a normaltissue saturating dose will have little effect on the delivery of theconjugated antibody to the tumor. The major considerations aresaturation of the more accessible tumor sites and affinity differencesbetween unconjugated, "cold" antibody and the conjugate. If theconjugate has substantially less affinity, too large a dose ofunconjugated antibody will compete off the conjugate and thus efficacy-- therapeutic or detection - will be reduced.

A second method for reducing cross-reactivity with normal tissues thatcontain the cross-reactive associated antigen involves directed prior orco-administration of unconjugated antibody (see Example III). Generally,venous or arterial access is achieved through percutaneouscatheterization. For example, the renal arteries or veins may becatheterized to perfuse the kidneys selectively. Alternatively, acatheter may be placed into an appropriate vessel (e.g., hepatic arttryfor liver) at he time of surgery. The unconjugated antibody is injectedby the catheter to contact the antigen sites of the normal organ withthe "first pass" of antibody. The antibody may be whole or fragmented.Either simultaneously or thereafter, the conjugated antibody is injectedinto the mammal's general circulation. Binding of the specificconjugated antibody to the previously perfused sites in the normalorgans is reduced, thus increasing the availability of the conjugatedspecific antibody for the target cells. This also results in reducedtoxicity to the cross-reactive, non-target organ by the specificconjugated antibody.

A third method involves either peripheral vein or directed infusion asdescribed above. Instead of using unconjugated antibody as the blockingantibody, a conjugate of antibody and detoxified cytotoxin or BRM isinfused where the detoxification (i.e., reduced toxicity) processpreserves the sites of recognition and/or binding by normal cells (seeExample IV). In a preferred embodiment, the detoxified agent is eitherfree or bound to the nonspecific antibody, and the detoxified conjugateis injected prior to the administration of specific antibody conjugateand, if necessary, may be administered as a constant infusion.

In another method, where the tumor to be treated is localized in anorgan or a limb, conjugated antibody is injected via directed catheterinto the organ or limb following peripheral blockage of cross-reactiveand nonspecific sites with unconjugated specific and/or deeoxified,conjugated irrelevant and/or unconjugated irrelevant antibody.

An additional method provides for injection of unconjugated specificantibody via catheter or other means of directing the firstadministration of the antibody to a defined organ that contains thecross-reactive antigen where the tumor to be treated is disseminatedoutside of the organ. Unconjugated irrelevant antibody and/ordetoxified, conjugated, nonspecific antibody are injected by peripheralvein. Conjugated specific antibody is then injected intraveneously.

3. Reducing Antiglobulin Responses Directed at the Specific Antibody

Irrelevant antibody is administered prior to or simultaneously withspecific antibody or specific antibody conjugate. The irrelevantantibody may be administered in higher doses than the specific antibody.In one preferred embodiment, the irrelevant antibody is wholeimmunoglobulin and the specific antibody is an antibody fragment (seeExample V). In another preferred embodiment, the ratio of specific tononspecific antibody ranges from about 1:1 to about 1:100 and ispreferably 1:5. In another preferred embodiment, the nonspecificantibody is whole immunoglobulin and the specific antibody is Fab or Fv.The basis for this strategy is that the higher dose and use of whole,not fragment, of the irrelevant antibody is more likely to evoke animmunological response than the lower dose and less immunogenicfragments of specific antibody.

When a patient develops antiglobulins that bind to the irrelevantantibody but not the specific antibody, the same irrelevant antibody maybe administered in a subsequent dose ahead of specific antibody toadsorb the circulating antiglobulin and in higher doses to blocknonspecific sites in normal tissues. Any antiglobulins to irrelevantantibody that cross-react with specific antibody would be complexed anddeposited in the kidney or reticuloendothelial system, depending on thesize of the complex. Most importantly, conjugated specific antibodygiven subsequently would not be complexed, but would be free to bind totarget cells. Alternatively, a second irrelevant antibody that is notrecognized by the antiglobulin response may be substituted in subsequentinjections. A combination of irrelevant antibodies may also be used.

EXAMPLES I. Reducing Nonspecific Uptake

Monoclonal antibody (Mab) NR-2AD is a murine IgG_(2a) immunoglobulinthat was designed as an anti-idiotype that bound to a single patient'sB-cell lymphoma and to no other human tissue. MAb 9.2.27 is a murineIgG_(2a) antibody that recognizes the 250 Kilodaltonglycoprotein/proteoglycan melanoma-associated antigen. Both were scaledup by in vitro cell culture, purified by column chromatography, andtested for purity and sterility to meet the draft guidelines forinjectable monoclonal antibodies from the Office of Biologics, Food &Drug Administration ("Points to consider in the manufacture ofinjectable monoclonal antibody products intended for human use in vivo:Revised draft of 6/11/84"). MAb 9.2.27 was digested with pepsin and theF(ab)'₂ fragment purified from residual intact antibody.

NR-2AD 50 mg was diluted in normal saline and injected intravenouslyinto patient 8501.08 who had metastatic malignant melanoma. One hourlater, 2.5 mg Tc-99 m labeled 9.2.27 F(ab)'₂ was administeredintravenously. The patient's blood was withdrawn and counted with aTc-99 m standard. Surprisingly, the serum half-life (t1/2) of thelabeled F(ab)'₂ was 17 hours. The average serum t1/2 of the labeled9.2.27 F(ab)'₂ for patients receiving NR-2AD was 12 hours compared to 6hours for those who did not receive NR-2AD prior to the labeledfragment. The patient's tumor was visualized by gamma camera imaging andthen excised. Another unexpected result was the detection of a tumorthat weighed only 0.25 gm.

II. Reducing Cross-Reactive Binding of Specific Antibody Conjugates A.Cold-specific antibody blocks uptake of conjugated specific antibody innormal organs

The effect of reducing cross-reactive uptake of a radiolabeledmonoclonal antibody preparation was assessed in the context of adiagnostic imaging clinical trial. The patient {#8501.22) examined inthe study was a 32-year-old male with a metastatic melanoma lesion inthe left posterior portion of the neck. The lesion consisted of atumorinvolved lymph node measuring 2×2 cm.

The patient received two schedules of radiolabeled antibody three daysapart. The first schedule of antibody consisted of two doses: a 50 mgdose of non-radiolabeled intact irrelevant antibody (NR-2AD)administered intravenously followed 1 hour later by a 2.5 mg dose ofTc-99 m radiolabeled Fab fragment of a MAb 9.2.27. The localization ofradiolabel within the patient was assessed by gamma camera imaging overa 7 hour period. The second schedule was identical, except that 7.5 mgof non-radiolabeled F(ab)'₂ fragment of 9.2.27 was administered 5minutes prior to the radiolabeled Fab preparation. The 50 mg dose ofintact irrelevant antibody was administered in both schedules, in orderto reduce nonspecific uptake of the radiolabeled antibody into normaltissues. The non-radiolabeled target specific F(ab')₂ preparation wasadministered for the purpose of reducing cross-reactive uptake of theradiolabeled antibody by non-target tissues.

The results of the study showed that omission of the non-radiolabeledtarget-specific antibody (9.2.27 F(ab')₂ was accompanied at 7 hourspost-infusion by uptake of radiolabeled antibody into non-target tissue,specifically spleen, bone marrow and kidney. The known tumor site wasnot imaged. Pre-infusion of the non-radiolabeled target-specificantibody (9.2.27 F(ab')₂ was accompanied at the same time point byuptake in the kidney, but no demonstrable uptake in the spleen, bonemarrow or other normal organs. This result was unexpected since themarrow and spleen uptake had previously been assumed to be nonspecific.In addition, the known tumor in the neck was clearly visible, confirmingthat blocking cross-reactive binding sites by prior infusion ofnon-radiolabeled target-specific antibody both reduced non-target tissuelocalization and increased tumor localization of the radiolabeledtarget-specific antibody.

B. Cold-Specific Antibody Blocks Uptake of the Labeled Specific Antibodyby an Epitope-Specific Mechanism

Patient 8501.29 was a 49-year-old Caucasian male who presented withmelanoma on his back that then spread to lymph nodes under his arm. Atthe time of imaging with the 99 m_(Tc) -labeled antibodies, the patienthad presumed disease in the right axilla (recurrent), questionable smallsubcutaneous metastases on his arm and back, and diffuse hepaticinvolvement, as evidenced by CT scan and elevated hepatic transaminases.

The purpose of the test in this patient was to determine if thecold-specific antibody blocked normal tissue accumulation of the labeledantibody in an antigen-binding site (i.e., epitope-specific) manner. Twoantibodies, NR-ML-05 and 9.2.27, that each recognized distinct epitopesof the 250 Kd. glycoprotein/proteoglycan melanomaassociated antigen werechosen for this study.

For the first procedure, the patient received irrelevant antibody NR-2AD(NRX 900.00), cold-specific 9.2.27 (NRX-112), and then radiolabeledNR-ML-05 (NRX 118.03) so that the cold-specific blocker and the labeledantibody recognized distinct epitopes of the same antigen. Gamma cameraimaging 4 and 8 hours after injection revealed hepatic metastases, anaxillary chain of nodes, but also images of marrow (sternum and pelvis)and spleen. A repeat procedure was identical but substituted NR-ML-05(NRX 118) as the cold-specific blocker for the 9.2.27 used in the firstprocedure. Gamma camera imaging revealed the same sites of disease, butthis time with the absence of marrow (sternum and pelvis) and splenicuptake of the label.

This example, using the same patient as his own control, demonstratedconclusively that the cold-specific antibody blocked uptake of thelabeled specific antibody into normal organs in an epitope-specificmanner.

III. Directed Infusion to Reduce Cross-Reactive Binding

Percutaneous catheterization of the celiac and/or pancreatoduodenalarteries is performed through the femoral artery. MAb NR-CO-1 thatreacts with colon carcinoma and cross-reacts with normal pancreas islabeled with Tc-99 m. Unlabeled NR-CO-1 F(ab)'₂ fragments (10 mg) areinjected by 10 minute infusion through the catheter followed by a normalsaline flush (directed infusion). At the end of the directed infusion,the labeled NR-CO-1 (2.5 mg) is injected by peripheral vein. Omittingthe directed infusion results in increased localization in the pancreasof the subsequently administered, labeled antibody, indicating that thedirected infusion can selectively reduce binding of conjugates in organscontaining cross-reactive antigen.

IV. Reducing Nonspecific Binding of Conjugates

Pseudomonas exotoxin (PE) and diphtheria toxin (DT) are bacterialproteins that interfere with protein synthesis in cells byADP-ribosylation of elongation factor-2 (EF-2). DT binds to nicotinamideat glutamic acid in position 148 in the molecule. Substitution ofaspartic acid in this position (DT_(ASP)) reduces the activity of DT to1% of the native molecule, but does not affect its binding to cells. PEis thought to have a "pocket" for binding to its substrate that containsa tyrosine (position 481) and glutamic acid (position 553). Iodinationof tyrosine 481 (PE_(tyrI)) is expected to result in >50% loss inactivity of PE (PE_(tyrI)) but retention of its binding ability.

NR-2AD (irrelevant immunoglobulin) is reacted with 25 mM dithiothreitoland PE_(tryI) or DT_(ASP) with SMPB [succinimidyl(4-p-maleimidophenyl)butyrate], and the unreacted reagents are removed. The derivatizedantibody and toxin are mixed together, producing NR-2AD-S-C-PE_(tryI) orNR-2AD-S-C-DT_(ASP) (detoxified conjugates) that are purified by columnchromatography. 20-500 mg of the detoxified conjugates is administeredintraveneously prior to administration of NR-ML-05-S-C-PE orNR-ML-05-S-C-DT (specific toxin conjugates). NR-ML-05 is a murinemonoclonal antibody that recognizes a 250 kilodaltonglycoprotein/proteoglycan human melanoma-associated antigen.

The prior administration of the detoxified conjugates is expected toreduce specific conjugate binding in normal nonspecific sites(nonspecific has two components in this example, viz, the nonspecificbinding of the irrelevant immunoglobulin part of the conjugate and thenonspecific binding of the modified toxin). The specific toxin conjugateis then injected intravenously in doses two times or greater than couldbe given without the blockade of non-specific sites with detoxifiedconjugates. Additionally, unconjugated NR-2AD and unconjugated NR-ML-05are given prior to the specific toxin conjugates. These methods permitthe administration of higher doses of potent immunoconjugate forincreased localization and improved tumor reduction.

V. Reducing Antiglobulin Response Directed at a Specific Antibody

The effect of co-administration of an irrelevant monoclonal antibody(NR-2AD) on subsequent antiglobulin development to a specific MAb(9.2.27) was assessed in the context of a diagnostic imaging trail.Sixteen patients with metastatic malignant melanoma received 1 to 25 mgdoses of Tc-99 m radiolabeled 9.2.27 monoclonal antibody or fragmentsthereof, preceded by 5 minutes by doses of 7.5 to 9 mg ofnon-radiolabeled 9.2.27 or fragments thereof. Thus, patients receivedtotal doses of 10 mg of the 9.2.27 antibody and its fragments. One hourprior to administration of the specific antibody, 50 mg doses of anon-radiolabeled intact irrelevant antibody (NR-2AD) was administered to11 of the 16 patients.

Serologic evidence of an antiglobulin response was evaluated using asolid-phase, enzyme-linked immunoassay (ELISA) employing either thetarget-specific (9.2.27) or irrelevant (NR-2AD) antibody as the captureantigen. Serum specimens were evaluated from time points ranging from 2weeks to over 6 months following treatment. Six of the eleven patientsevaluated receiving NR-2AD demonstrated evidence of an antiglobulinresponse to the irrelevant NR-2AD antibody of greater magnitude thanthat which was obtained in the upper 95th percentile of 60 healthycontrols. In the same group of eleven patients, only one persondemonstrated an antiglobulin response to the target-specific 9.2.27antibody above the 95th percentile of the healthy control population.

These data indicate that prior administration of a 5-fold excess of anirrelevant MAb preparation resulted in antiglobulin responses, in thoseindividuals who developed an antiglobulin response, which was skewedtoward the irrelevant rather than the target-specific MAb.

VI. Repeated Administration of Antibody in the Presence of Antiglobulins

Patient #8501.08 in the 99 m_(Tc) imaging trail of melanoma received twoimaging procedures within a one-week period with the 9.2.27melanoma-specific antibody and the NR-2AD irrelevant antibody. Thepatient returned after 8 months and was repeat imaged with the NR-ML-05melanomaspecific antibody and the NR-2AD antibody. At that time, thepatient had developed a 19-fold increase in antiglobulin directed to theNR-2AD antibody. After premedication with 50 mg diphenhydramine, thepatient received NR-2AD 41 mg diluted in normal saline slowly over 60minutes. He then received 10 mg NR-ML-05 cold-specific antibody followedby NR-ML-05 Fab labeled with 99 m_(Tc). The labeled antibody showed noevidence of altered biodistribution and successfully targeted knownsubcutaneous and liver tumors.

By contrast, studies in normal guinea pigs immunized with murinemonoclonal antibodies and imaged with either the same or an irrelevant⁹⁹ m Tc-labeled antibody demonstrated that the presence of specificantiglobulin altered biodistribution of labeled antibody. Administrationof a radiolabeled antibody to which antiglobulin was reactive resultedin biodistribution of the radiolabel to liver and spleen. If thecirculating antiglobulin was not specific for the radiolabeled antibody,then biodistribution was indistinguishable from biodistribution in thenon-immunized animal.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

We claim:
 1. A method of enhancing delivery to solid tumor target cellswithin a mammal of conjugated specific antibodies or fragments thereofpharmaceutically active and specific for said target cells, comprisingthe steps of:administering to said mammal an adequate dosage of blockingantibodies or fragments thereof, said blocking antibodies being capableof blocking the binding of the conjugated specific antibodies orfragments thereof to non-target tissue(s) through antigen recognition;and then administering to said mammal a diagnostically ortherapeutically effective dosage of said conjugated specific antibodiesor fragments thereof, said conjugated specific antibodies being specificfor said solid tumor target cells.
 2. The method of claim 1 wherein saidblocking antibodies or fragments thereof are capable of cross-reactive,epitope-specific binding to non-target cells.
 3. The method of claim 1wherein said antibody fragments are selected from the group consistingof F(ab)', F(ab)'₂, Fab, Fv, and mixtures thereof.
 4. The method ofclaim 1 wherein said solid tumor target cells are characterized byhaving tumor-associated antigen.
 5. The method of claim 1 wherein any ofthe antibodies are monoclonal antibodies.
 6. The method of claim 1wherein any of the antibodies are polyclonal antibodies.
 7. The methodof claim 1 wherein the conjugated specific antibodies compriseantibodies or fragments thereof specific for said target cellsconjugated to a cytotoxin.
 8. The method of claim 1 wherein theconjugated specific antibodies comprise antibodies or fragments thereofspecific for said target cells conjugated to a radionuclide.
 9. Themethod of claim 1 wherein the effective dosage of conjugated specificantibodies or fragments thereof is diagnostically effective.
 10. Themethod of claim 1 wherein the effective dosage of conjugated specificantibodies or fragments thereof is therapeutically effective.
 11. Themethod of claim 1 wherein the mammal is a human.
 12. A method ofenhancing the localization at a solid tumor target site of conjugatedspecific antibodies or fragments thereof specific for an antigencontained on the target site and also on a non-target tissue or organwithin a mammal, comprising the steps of:perfusing said non-targettissue or organ with an adequate dosage of blocking antibodies orfragments thereof, said blocking antibodies being capable of blockingthe binding of the conjugated specific antibodies or fragments thereofto non-target tissue(s) or organ through antigen recognition; andadministering to the mammal a diagnostically or therapeuticallyeffective dosage of said conjugated specific antibodies or fragmentsthereof specific for said antigen.
 13. The method of claim 12 whereinsaid blocking antibodies or fragments thereof are capable ofcross-reactive, epitope-specific binding to the non-target tissue ororgan.
 14. The method of claim 12 wherein said antibody fragments areselected from the group consisting of F(ab)', F(ab)'₂, Fab, Fv, andmixtures thereof.
 15. The method of claim 12 wherein said tissue ororgan is characterized by having tumor-associated antigen.
 16. Themethod of claim 12 wherein the antibodies are monoclonal antibodies. 17.The method of claim 12 wherein the antibodies are polyclonal antibodies.18. The method of claim 12 wherein the conjugated specific antibodiescomprise antibodies or fragments thereof conjugated to a cytotoxin ordrug.
 19. The method of claim 12 wherein the conjugated specificantibodies comprise antibodies or fragments thereof conjugated to aradionuclide.
 20. The method of claim 12 wherein the blocking antibodiesor fragments thereof and the conjugated specific antibodies or fragmentsthereof are administered simultaneously.
 21. The method of claim 12wherein the blocking antibodies or fragments thereof are administeredprior to the conjugated specific antibodies or fragments thereof. 22.The method of claim 12 wherein the effective dosage of said conjugatedspecific antibodies or fragments thereof is diagnostically effective.23. The method of claim 12 wherein the effective dosage of saidconjugated specific antibodies or fragments thereof is therapeuticallyeffective.
 24. The method of claim 12 wherein the mammal is a human. 25.A method of targeting melanoma in humans, comprising the stepsof:administering to said human an adequate dosage of unlabeled specificantibody or fragment thereof that binds to a melanoma-associatedantigen; and then administering to said human a diagnostically ortherapeutically effective dose of labeled specific antibody or fragmentthereof that binds to the same epitope of the melanoma-associatedantigen as the unlabeled specific antibody.
 26. The method of claim 25wherein the antibody or fragment thereof that binds to amelanoma-associated antigen recognizes the 250 Kd.glycoprotein/proteoglycan.
 27. The method of claim 25 wherein theantigen-binding region of the antibody or fragment thereof is selectedfrom the group consisting of antigen-binding regions of antibodies9.2.27 and NR-ML-05, and their clones, chimaeras and derivatives. 28.The method of claim 25 wherein the antigen-binding region of theantibody or fragment thereof recognizes the G_(D3) glycolipidmelanoma-associated antigen.
 29. The method of claim 25 wherein theantigen-binding region of antibody or fragment thereof recognizes theP97 melamona-associated antigen.
 30. A method of enhancing delivery tosolid tumor target cells within a mammal of conjugated specificantibodies or fragments thereof pharmaceutically active and specific forsaid target cells, comprising the steps of:administering to said mammalan adequate dosage of blocking antibodies or fragments thereof, whereinsaid blocking antibodies are the unconjugated form of the conjugatedspecific antibodies or fragments thereof; and then administering to saidmammal a diagnostically or therapeutically effective dosage of saidconjugated specific antibodies or fragments thereof, said conjugatedspecific antibodies being specific for said solid tumor target cells.31. The method of claim 30 wherein the conjugated specific antibodiescomprise antibodies or fragments thereof specific for said target cellsconjugated to a radionuclide.